• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1699-1706
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• 2014

A lichenase gene (mt-lic) was identified for the first time through function-based screening of a soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of homology with endo-${\beta}$-1,3-1,4-glucanase (having both lichenase and chitosanase activities), encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed and purified from Escherichia coli was able to efficiently hydrolyze both barley ${\beta}$-glucan and lichenan. The enzyme showed maximal activity at a pH of 6.0 at $50^{\circ}C$, with Azo-barley-glucan as the substrate. The metal ions $Mn^{2+}$, $Mg^{2+}$, $Ca^{2+}$, and $Fe^{2+}$ enhanced the enzymatic activity, whereas the $Cu^{2+}$ and $Zn^{2+}$ ions inhibited the enzymatic activity. The $K_m$ and $V_{max}$ values of the purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein, respectively.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.80-80
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• 1995

T4 Endonuclease V (Mw 16,000) acts as a repair enzyme for UV induced pyrimidine dimers in DNA. Many researchers have studied the biochemical characteristics of the enzyme. However the precise action mechanism of T4 endo V has not fully elucidated yet. In our laboratory NMR spectroscopy technique is being used for the structural study of T4 endo V. Because of its low temperature stability and high content of ${\alpha}$-helix, the conventional $^1$H NMR technique was inapplicable. Therefore we utilized stable isotope labeling technique and so far prepared about 10 amino acid specific labeled proteins. The HSQC spectra of amino acid specific labeled proteins will help us to interpret the triple resonance 3D, 4D data which are under processing, We also studied the behaviors of specific amino acid residues whose roles might be critical. When the enzyme labeled by $\^$15/N-Thr was mixed with the substrate oligonucleotide (semispecific -TT- sequence), one crosspeak in its HSQC spectrum was completely desappeared, which means that one of seven Thr residues is in the binding site of the enzyme with DNA, This result is well consistent with previous report that implicated the Thr 2 residue in the activity of the enzyme. Similar studies were carried on the behaviors of Arg and Tyr residues.

• Journal of Life Science
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• v.22 no.12
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• pp.1724-1728
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• 2012

A marine bacterial strain that degraded fucoidan from Ecklonia cava was isolated from seawater. The crude fucoidanase of this strain efficiently degraded fucoidan at pH 8 and $50^{\circ}C$. The crude enzyme hydrolyzed 7.1% (w/w) fucoidan within 24 hrs from an 1% (w/v) fucoidan solution and produced oligosaccharides by endo-type hydrolysis as the reaction products. The results of 16S rRNA gene sequence analysis and biochemical tests permitted a tentative identification of strain SB 1493 as a Pseudoalteromonas species.

• Microbiology and Biotechnology Letters
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• v.28 no.4
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• pp.195-201
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• 2000

Glucoamylase of Saccharomyces diastaticus is produced as a large precursor composed of signal peptide (21 amino acid residues), Thr and Ser-rich region and functional glucoamylase. To evaluate the utility of the glucoamylase signal peptide (GSP) for the secretion of foreign proteins, four types of GSP mutants (ml : Pro-18 longrightarrowLeu-18, m2 : Tyr-13 longrightarrowLeu, m3 : Ser-9longrightarrowLeu-9, m4 : Asn-5 longrightarrowPro-5) were constructed and secretion efficiency of each mutant was compared with that of native GSP by the expression and secretion of Bacillus subtilis CMCase under the control of GAP in N-terminal domain and hydrophobic domain. n mutant 4, a polar amino acid was replaced by a helix - breaking Pro residue. CMCase activity assay and Western blot analysis revealed that CMCase secretion by GSP mutants replaced by Leu were increased compared with native GSP. In the case of m2 and m3, the substitution of Leu for Tyr-13 and Ser-9 in the hydrophobic region resulted in a twofold increase in the extracellular CMCase activity.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.58-66
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• 2007

Isolation, expression, and characterization of a novel $endo-{\beta}-1,3(4)-D-glucanase$ with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a ${\beta}-glucanase$ protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other ${\beta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${\beta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{\beta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{\circ}C$. The $K_m,\;V_{max},\;and\;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{\mu}mol/min{\cdot}mg,\;and\;3,131s^{-1}$, respectively. For barley ${\beta}-glucan$ the values are 3.73mg/ml, $8,939{\mu}mol/min{\cdot}mg,\;and\;4,026s^{-1}$, respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.559-563
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• 2000

A dextransucrase gene (dsrB742) that expresses a dextransucrase to synthesize mostly ${\alpha}-1{\rightarrow}6$ linked dextran with a low amount (3-5%) of ${\alpha}-1{\rightarrow}3$ branching was cloned and sequenced from Leuconostoc mesenteroides B-742CB. The 6.1-kb PstI fragments were ligated with pGEM-3Zf(-) and transformed into E. coli $DH5{\alpha}$. The recombinant clone (pDSRB742) synthesized dextran on an agar plate containing 2% (w/v) sucrose. The dextran synthesized was hydrolyzed with Penicillium endo-dextranase. The hydrolyzate was composed of glucose, isomaltose, isomaltotriose, and branced pentasaccharide. The nucleotide sequence of dsrB742 showed one open reading frame (ORF) composed of 4,524 bp encoding dextrasnsucrase. The deduced amino acid sequence revealed a calculated molecular mass of 168.6 kDa. It also showed an activity band of 184 kKa on a non-denaturing SDS-PAGE (10%). The amino acid sequence of DSRB742 exhibited a 50% similarity with DSRA from L. mesenteroides B-1299, a 70% similarity with DSRS from L. mesenteroides B-512 (F, FMCM) and a 45-56% similarity with Streptococcal GTFs.

• Korean Journal of Microbiology
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• v.31 no.3
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• pp.203-207
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• 1993

Five different secretion vectors were constructed by varying the signal sequences and .alpha.-antitrypsin (.alpha.1-AT) a numan secretory protein, was produced from yeast cells. The signal sequences used are those of acid phosphatase (PH05) and .alpha.-factor (M f.alphal1) of Saccharomyces cerevisiae, glucoamylase (STA1) of Saccharomyces diastaticus, and human .alpha.1-AT. Four vectors directed the efficient secretion of .alpha.1-AT ito the culture media. The secretion vector carrying the glucoamylase signal sequence (pGAT11) showed the highest efficiency of secretion. About 70% of .alpha.1-AT produce dwere secreted into the media. The endo H treatment of partially purified .alpha.1-AT indicates that the secreted .alpha.1-AT appeared to be glycosylated. This glycosylation pattern was altered when amino acid substitution mutations were introduced at the three glycosylation sites of .alpha.-AT.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.800-805
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• 2007

Two endoglucanases with processive cellulase activities, produced from Fomitopsis palustris grown on 2% microcrystalline cellulose(Avicel), were purified to homogeneity by anion-exchange and gel filtration column chromatography systems. SDS-PAGE analysis indicated that the molecular masses of the purified enzymes were 47 kDa and 35 kDa, respectively. The amino acid sequence analysis of the 47-kDa protein(EG47) showed a sequence similarity with fungal glycoside hydrolase family 5 endoglucanase from the white-rot fungus Phanerochaete chrysosporium. N-terminal and internal amino acid sequences of the 35-kDa protein(EG35), however, had no homology with any other glycosylhydrolases, although the enzyme had high specific activity against carboxymethyl cellulose, which is a typical substrate for endoglucanases. The initial rate of Avicel hydrolysis by EG35 was relatively fast for 48 h, and the amount of soluble reducing sugar released after 96 h was $100{\mu}g/ml$. Although EG47 also hydrolyzed Avicel, the hydrolysis rate was lower than that of EG35. Thin layer chromatography analysis of the hydrolysis products released from Avicel indicated that the main product was cellobiose, suggesting that the brown-rot fungus possesses processive EGs capable of degrading crystalline cellulose.

• BMB Reports
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• v.44 no.1
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• pp.52-57
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• 2011

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and $37^{\circ}C$, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.

• Mycobiology
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• v.46 no.2
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• pp.168-171
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• 2018

In 2016, a severe leaf spot disease was found on Iris ensata Thumb. in Nanjing, China. The symptom was elliptical, fusiform, or irregularly necrotic lesion surrounded by a yellow halo, from which a small-spored Alternaria species was isolated. The fungus was identified as Alternaria iridiaustralis based on morphological characteristics. The pathogenicity tests revealed that the fungus was the causal pathogen of the disease. Phylogenic analyses using sequences of ITS, gpd, endoPG, and RPB2 genes confirmed the morphological identification. This study is the first report of A. iridiaustralis causing leaf spots on I. ensata in China.